Polyphenols are those compounds which comprise more than one phenolic group. Among the polyphenols are the following classes: flavonoids (a term often used to denote polyphenols in general, but more commonly in Europe to denote only the flavones), the flavanols, proanthocyanidins (also called procyanidols, procyanins, procyanidins and tannins) and anthocyanins.
The flavones are compounds with a basis structure shown in FIG. 1 in which two benzene rings (A and B) are linked with a heterocyclic six member ring C containing a carbonyl ring. Ring B can be joined in position 2 (as illustrated) to give a flavone or to position 3 to give an iso flavone. Hydroxylation can occur at positions 3, 5, 7, and 3', 4', 5' to give compounds called flavonols. Typical examples of flavonols are: quercetin (hydroxylated at positions 3, 5, 7, 3', 4'), kaempferol (hydroxylated at positions 3, 5, 7, 4') and myricetin (hydroxylated at positions 3, 5, 7, 3', 4', 5'). They can exist naturally as the aglycone or as O-glycosides (e.g. D-glucose, galactose, arabinose, rhamnose, etc). Other forms of substitution such as methylation, sulphation and malonylation are also found.
The flavanols have a basic structure shown in FIG. 2. The two most common flavanols are catechin (hydroxyl groups positions 5, 7, 3', 4') and its stereo-isomer epicatechin. The hydroxyl groups can be esterified with gallic acid. The proanthocyanidins are polymers of catechin and/or epicatechin and can contain up to 8 units or more. These compounds are often called proanthocyanidins, procyanidins or tannins.
The anthocyanins are colored substances, sometimes called anthocyanidins. Typical examples are: cyanidin (hydroxylated at positions 3, 5, 7, 3', 4'), delphinidin (hydroxylated at positions 3, 5, 7, 4', 5') and pelargonidin (hydroxylated at positions 3, 5, 7, 3'). The hydroxyl groups are usually glycosylated and/or methoxylated (e.g. malvidin at 3', 5').
Within the general term "polyphenols" are included the dihydroxy-or-tri-hydroxy benzoic acids and the phytoalexins, a typical example of which is resveratrol.
Polyphenols are found in various amounts in large numbers of natural products especially plant material such as fruit and vegetables. A particular rich source are grapes, in which the polyphenols are plentiful in the skins and seeds, but not in the pulp. During the manufacture of grape juice, quantities of polyphenols are expressed into the juice, and the polyphenol content will depend on the type of grape, the climate in which it is grown, and the manufacturing process used in making the juice. Some grape juice, especially that made from the Concord grape, may contain as much as 2.5 g/L polyphenol. Grape skins and seeds are commercially extracted with water and other solvents to obtain polyphenols. In particular polyphenols from grape skins and seeds become incorporated into wine during the vinification process. Red wine is made by maintaining contact between the fermenting liquor and the crushed grape residue (pomace) for prolonged periods, while in the manufacture of white wine the grape skins are removed relatively quickly. Accordingly, wine in general, and red wine in particular, contains reasonable amounts of polyphenols, amounting to about 1-3 g/L and is thus a potential commercial source of polyphenolic compounds.
Polyphenols are known to have antioxidant properties and have potential use in the food, cosmetic and pharmaceutical industries. However, in order for polyphenols to be used commercially the polyphenols have to be separated from grape extracts and wine in a more concentrated form. Dealcoholised wine has been available for many years and can be concentrated by distillation. Also it is possible under certain conditions to obtain a dry power from dealcoholised wine by spray drying.
Among the polyphenols, the flavonols have been shown to have many useful properties as antioxidants, and to decrease platelet stickiness. Epidemiological studies have shown that countries and people with a high flavonol intake have less coronary heart disease (Hertog M G L et al., 1995, Arch Int Med 155, 381-6).
Flavonols are present in grapes and values ranging from 8 to 97 mg/Kg FW have been reported (Macheix et al., 1990 Fruit Phenolics pp. 378 CRC Press Boca Raton). In grape skins the flavonols occur as the glycosides and the free aglycone is not present. During the fermentation process, some of the sugar is split off and the aglycone formed. On average about 50% of the flavonol exists in wine as the aglycone. It has been discovered that the flavonol content of grape skins and wine is very variable and depends on the variety of grape and more especially the amount of sunshine in which the grapes are grown. The flavonols have a yellow color and act as filters to blue and ultraviolet light which is very injurious to the grape. During period of intense sunlight more flavonols are synthesized to protect the grape, and consequently the grape skins and the wine from which it is made has a high concentration of flavonols. (Price S F et al., Am J. Enol Vitic 46 187-194, 1995).
Some wines in France have only 5 mg/L flavonol (calculated as aglycone) whereas up to 150 mg/L have been reported in some Californian wines. The flavonols are virtually absent from the pulp and grape seeds and only trace quantities are present in commercial anthocyanin powders extracted from pomace after making red wine.
Wine contains many substances besides alcohol and polyphenols; these may include carbohydrates, especially glucose, tartaric acid and mineral salts. A typical red wine contains about 23 g/L solids and 1.5 g/L polyphenols. For commercial use it would be preferable that the polyphenols should be in a stable and easily transportable form and more preferably as a solid. In solution, especially in dealcoholised wine, the polyphenols are capable of being oxidized, and contaminated with micro-organisms. A method is therefore needed to isolate the total polyphenols in wine from the other constituents.
Although grape juice is often less rich in polyphenols than wine, it is a readily available commodity, and can be used as a source of polyphenols. It has a high sugar content and a high content of solids.
It would also be a great advantage to use, as a source of polyphenols, by-products of the wine-making process such as grape skins or wine pomace (a mixture of skins and seeds obtained after pressing the grapes to obtain the juice). Pomace contains grape seeds which have been used extensively in the industry to obtain grape seed oil, and polyphenols. Two such products containing polyphenols are Endotenol.TM. (Sanofi-Labaz, France) and Activin.TM. (Interhealth Nutritionals Inc., California, USA).
Grape seeds contain chiefly one class of polyphenols, the proanthocyanidins, with catechin, epcatechin and their esters as minor components. The material obtained from grape seed does not have such a wide spectrum of polyphenols as are present in wine or grape skins. Grape seed extract is unsuitable for preparing a "total phenolic pool" and does not protect low density lipoproteins from oxidation in vivo.
Grape skins before fermentation contain more of the flavonols present in the whole grape. When red wine is made flavonols are leached out of the skins and seeds by the aqueous ethanol and the pomace will be depleted of some of the flavonols. In white wine manufacture, the skins and seeds are not usually present during the fermentation process and most of the flavonols are left in the pomace.
Those skilled in the art can predict that to develop a manufacturing procedure will not be straightforward. Presented with a complex mixture of organic and inorganic chemical as in grape juice, wine, grape skins and pomace the possibility of obtaining a high concentration of polyphenols and flavonols initially present as only about 5-10% and 0.1-0.2% of the total solids respectively represents a challenge which would need inventive skills. Further, the desirability of developing such a process would not be obvious.
Polyphenols are a mixture of substances of very different molecular weight and polarity. They are known to be soluble or partly soluble in polar solvents or admixtures thereof (e.g., water, ethanol, methanol, acetone, ethyl acetate). Also certain resins such as Sephadex LH-20 have been used for the purpose of separating polyphenols for analysis by thin layer, column and HPL chromatography.
Various methods have been developed to extract polyphenols from grape skins, especially from red or black grapes since the anthocyanin pigment is used as a colorant in foods. The methods so far disclosed have not been directed to obtaining a polyphenol extract with a high flavonol content. Flavonols are not present in grape seeds, and these have been extracted chiefly to obtain the proanthocyanidins.
Yokoyama et al. (U.S. Pat. No. 4,302,200) discloses a process for extracting anthocyanin-type color from natural products (grapes) which includes contacting the natural product with a sulfite ion-containing aqueous solution at a temperature of about 85.degree. C. or higher. Yokoyama et al. discloses a process suited to the recovery of anthocyanin, but not flavonols.
Hilton et al. (U.S. Pat. No. 4,320,009) discloses a process for obtaining anthocyanin pigment extracts in which large quantities of anthocyanin extract may be obtained from grape skin residue from wine fermentation. The latter would not contain the flavonols which are eluted from grape skins during the wine making process. In the method of Hilton et al. it is necessary to add filler such as maltodextrin before spray drying grape skin extracts. Such a method would give low concentration of polyphenols in the final composition.
Although Hilton et al. discloses a process for obtaining anthocyanins which includes preparing an aqueous grape skin extract, absorption of the pigments onto an ion exchange resin, elution from the resin, and if necessary, further chromatography on paper or a thin layer of silica gel, the process is entirely focused on obtaining anthocyanin and, as a result, flavonols would not be recovered.
Shrikhande (U.S. Pat. No. 4,452,822) discloses a process for improvements in the production of anthocyanin coloring material from red grape pomace or other anthocyanin sources using extraction with sulfur dioxide. The improvement is to treat the sulfur dioxide extract enzymatically to reduce or eliminate the solid material present in the extract. The method involves the use of H.sub.2 O.sub.2 which would oxidize and destroy flavonols.
Crosby et al. (U.S. Pat. No. 4,481,226) discloses a stabilized anthocyanin grape extract colorant. The stabilized product is made by combining tannic acid and anthocyanin grape extract colorant in an appropriate solvent, and recovering the product. The method is not directed to the recovery of flavonols.
Langston (U.S. Pat. No. 4,500,556) discloses a process for obtaining anthocyanin colorant by extraction from grape pomace. The method described in Langston is to contact grape pomace with HSO.sub.3 to form a complex. The complex is recovered by treating the liquid extract phase with a non-ionic adsorbent to adsorb the complex. The adsorbent bed is rinsed with water to remove the adsorbent water-soluble non-pigment material, such as sugar, organic acid and solid particles. The complex is then eluted from the adsorbent with an acidified organic solvent leaving the polymerized anthocyanin pigment behind. The preferred solvent is 100% ethanol acidified with a small amount of mineral acid which breaks the complex such that the anthocyanin free of HSO.sub.3 ions is eluted. The process is not directed to the recovery of flavonols, which would be destroyed therein.
Ford (U.S. Pat. No. 5,141,611) discloses a process for removing polyphenolic substances from a solution by adsorption on a polyamide resin having a specific porosity and extended surface. The process is not directed to the recovery of flavonols.
Frangi et al. (U.S. Pat. No. 5,484,594) discloses procyanidol oligomeric fractions extracted from vegetables and uses grape seeds as the starting material. The process does not include the isolation of flavonols which would be destroyed during the process if present.
Salagoity-Auguste et al. (J. Sci. Food and Agric. 1984 35 1241-1247) discloses an analytical process by which de-alcoholised wine was extracted with ethyl acetate which did not extract the anthocyanidins and gave a low yield of procyanidins. The extracts were removed from a column (C18 hydrocarbon polymer) with a solution containing perchloric acid. The method is not suitable for the preparation and isolation of polyphenols from grape extracts for human consumption.
Cheynier et al. (Am. J. Enol. Vitic. 1986 37 248-252) discloses a method whereby the skins were extracted with methanol and the solvent removed. The method used ethyl acetate to remove anthocyanins and the extract was then fractionated on a polyamide column.
Acetonitrile used in the chromatographic separation is an unsuitable solvent for food extraction methods and is not "Generally Recognized as Safe" (GRAS). Its use in food is not permitted.
Oszianski et al. (Am. J. Enol. Vitic. 1988 39 259-262) discloses a method of fractionation with is a Sep-Pak cartridge and also uses acetonitrile as a solvent.
Tomas-Barberan et al. (Phytochem. Anal. 1992, 3 178-181) uses synthetic mixtures of flavonols in an aqueous solution not grape extracts. Other polyphenols in grape extract were not studied or disclosed.
To summarize, many methods of extracting polyphenols have been developed but all of these have been directed towards obtaining anthocyanins, procyanidins or polyphenols other than flavonols, and/or are not suitable for preparing compositions for human consumption.
What is needed, therefore, is a method of extracting polyphenols which contain substantial amounts of flavonols and preparing compositions therefrom which are suitable for human consumption.